This invention relates to method and means for assaying collagenase in mammalian biologic fluids. More particularly, it relates to such method and means involving the use of collagen to quantitate the collagenase activity of gingival crevicular fluid, hereinafter referred to as GCF.
With the realization that standard clinical measurements of human periodontal disease are not indicative of current loss of clinical periodontal attachment or predictive of future loss of attachment (Haffajee, A. D., Socransky, S. S. and Goodson, J. M.: Clinical parameters or predictors of destructive periodontal disease activity, J. Clin. Periodontol. 10:257, 1983), attention has focused on the need for sensitive measures of active periodontal disease (Polson, A. M. and Goodson, J. M.: Periodontal diagnosis: current status and future needs, J. Periodontol. 56:25, 1985). Among the possible approaches for such a diagnostic test, biochemical analysis of GCF has received considerable attention (Fine, D. H. and Mandel, I. D.: Indicators of periodontal disease activity: an evaluation, J. Clin. Periodontol. 13:533, 1986). Study of the volume and constituents of the fluid that characterize periodontal health and disease has been conducted for almost 30 years.
Proteolytic enzymes are among the constituents of GCF that have been examined for their diagnostic potential. Among these, collagenase is of particular interest since collagen is the major structural protein of the periodontium, and loss of collagen is characteristic of both gingivitis and periodontitis (Narayanan, A. S. and Page, R. C.: Connective tissue of the periodontium: a summary of current work, Col. Rel. Res. 3:33, 1983.) Previous studies have correlated increased collagenase activity in GCF with increased gingival inflammation (Ohlsson, K., Olsson, I. and Tynelius-Bratthal, G.: Neutrophil leukocyte collagenase, elastase and serum protease inhibitors in human gingival crevices, Acta Odont. Scand. 31:51, 1973; Golub, L. M., Siegel, N., Ramamurthy, C. and Mandel, I.: Some characteristics of collagenase activity in gingival crevicular fluid and its relationship to gingival disease in humans, J. Dent. Res 55:1049, 1976). Furthermore, a study of beagle dogs with ligature-induced periodontitis indicated that collagenolytic activity in GCF was correlated with loss of attachment (Kryshtalskyj, E., Sodek, J. and Ferrier, J. M.: Correlation of collagenolytic enzymes and inhibitors in gingival crevicular fluid with clinical and microscopic changes in experimental periodontitis in the dog. Archs. Oral Biol. 31:21, 1986). Nevertheless, quantitation of collagenase activity in biologic fluids frequently involves the use of radio labelled collagen substrates (.sup.3 H-collagen, .sup.14C C-collagen) or gel electrophoresis to identify collagen breakdown fragments (Ohlsson et al., Ibid.; Golub et al., Ibid.; Kryshtalskyj et al., Ibid. These approaches cannot be conveniently incorporated into an in-office test kit to be used by the dentist or dental hygienist.
We have developed an approach to measuring collagenolytic activity in small volumes of mammalian biologic fluid. This test is based on degradation of Type I collagen that is bound to an inert surface, and is semi-quantitative by evaluating the zone of collagen clearance. The specificity of this reaction has been studied by examining the effect of different enzymes on the substrate. It has been determined that collagenase will rapidly degrade the film, trypsin will degrade the film only at very high enzyme concentrations and pepsin and various ground-substance degrading enzymes have essentially no effect on the film.